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1.
Biol Open ; 2019 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-31064740

RESUMO

Six2+ cap mesenchyme cells, also called nephron progenitor cells (NPC), are precursors of all epithelial cell types of the nephron, the filtering unit of the kidney. Current evidence indicates that perinatal "old" NPC have a greater tendency to exit the progenitor niche and differentiate into nascent nephrons than their embryonic "young" counterpart. Understanding the underpinnings of NPC development may offer insights to rejuvenate old NPC and expand the progenitor pool. Here, we compared the chromatin landscape of young and old NPC and found common features reflecting their shared lineage but also intrinsic differences in chromatin accessibility and enhancer landscape supporting the view that old NPC are epigenetically poised for differentiation. Annotation of open chromatin regions and active enhancers uncovered the transcription factor Bach2 as a potential link between the pro-renewal MAPK/AP1 and pro-differentiation Six2/b-catenin pathways that might be of critical importance in regulation of NPC fate. Our data provide the first glimpse of the dynamic chromatin landscape of NPC and serve as a platform for future studies of the impact of genetic or environmental perturbations on the epigenome of NPC.

2.
Development ; 145(10)2018 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-29712641

RESUMO

Nephron progenitor cells (NPCs) are Six2-positive metanephric mesenchyme cells, which undergo self-renewal and differentiation to give rise to nephrons until the end of nephrogenesis. Histone deacetylases (HDACs) are a group of epigenetic regulators that control cell fate, but their role in balancing NPC renewal and differentiation is unknown. Here, we report that NPC-specific deletion of Hdac1 and Hdac2 genes in mice results in early postnatal lethality owing to renal hypodysplasia and loss of NPCs. HDAC1/2 interact with the NPC renewal regulators Six2, Osr1 and Sall1, and are co-bound along with Six2 on the Six2 enhancer. Although the mutant NPCs differentiate into renal vesicles (RVs), Hdac1/2 mutant kidneys lack nascent nephrons or mature glomeruli, a phenocopy of Lhx1 mutants. Transcriptional profiling and network analysis identified disrupted expression of Lhx1 and its downstream genes, Dll1 and Hnf1a/4a, as key mediators of the renal phenotype. Finally, although HDAC1/2-deficient NPCs and RVs overexpress hyperacetylated p53, Trp53 deletion failed to rescue the renal dysgenesis. We conclude that the epigenetic regulators HDAC1 and HDAC2 control nephrogenesis via interactions with the transcriptional programs of nephron progenitors and renal vesicles.


Assuntos
Histona Desacetilase 1/genética , Histona Desacetilase 2/genética , Néfrons/embriologia , Organogênese/genética , Células-Tronco/citologia , Transcrição Genética/genética , Animais , Linhagem Celular , Proliferação de Células/genética , Células HEK293 , Fator 1-alfa Nuclear de Hepatócito/biossíntese , Fator 4 Nuclear de Hepatócito/biossíntese , Histona Desacetilase 1/metabolismo , Histona Desacetilase 2/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Nefropatias/genética , Proteínas com Homeodomínio LIM/genética , Camundongos , Camundongos Knockout , Néfrons/citologia , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ativação Transcricional/genética , Proteína Supressora de Tumor p53/genética
3.
Int Immunopharmacol ; 32: 55-61, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26800098

RESUMO

Pogostone, a major component of Pogostemon cablin, has been demonstrated to possess antibacterial, anti-fungal, immunosuppressive and anti-inflammatory properties. To investigate the potential therapeutic effect of pogostone on lipopolysaccharide (LPS)-induced acute lung injury (ALI), mice were pretreated with pogostone prior to LPS exposure. After LPS challenge, the lungs were excised and the histological changes, wet to dry weight ratios, MPO activity reflecting neutrophil infiltration, and MDA activity reflecting oxidative stress were examined. The inflammatory cytokines in the BALF were determined by ELISA assay. Moreover, the expressions of p65 and phosphorylated p65 subunit of NF-κB, and Nrf2 in the nucleus in lung tissues were measured by Western blot analysis, and meanwhile the dependent genes of NF-κB and Nrf2 were assessed by RT-qPCR. The results showed that pretreatment with pogostone markedly improved survival rate, attenuated the histological alterations in the lung, reduced the MPO and MDA levels, decreased the wet/dry weight ratio of lungs, down-regulated the level of pro-inflammatory mediators including TNF-a, IL-1ß and IL-6. Furthermore, pretreatment with pogostone enhanced the Nrf2 dependent genes including NQO-1, GCLC and HO-1 but suppressed NF-κB regulated genes including TNF-α, IL-1ß and IL-6. The mechanism behind the protective effect was correlated with its regulation on the balance between Keap1-Nrf2 and NF-κB signaling pathways. Therefore, pogostone may be considered as a potential therapeutic agent for preventing and treating ALI.


Assuntos
Lesão Pulmonar Aguda/tratamento farmacológico , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Óleos Voláteis/farmacologia , Óleos Voláteis/uso terapêutico , Lesão Pulmonar Aguda/imunologia , Lesão Pulmonar Aguda/patologia , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Animais , Líquido da Lavagem Broncoalveolar/imunologia , Citocinas/genética , Citocinas/imunologia , Proteínas do Citoesqueleto/imunologia , Proteína 1 Associada a ECH Semelhante a Kelch , Lipopolissacarídeos , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/patologia , Masculino , Malondialdeído/metabolismo , Camundongos , Fator 2 Relacionado a NF-E2/imunologia , NF-kappa B/imunologia , Peroxidase/metabolismo , Transdução de Sinais/efeitos dos fármacos
4.
J Am Heart Assoc ; 3(3): e000796, 2014 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-24847033

RESUMO

BACKGROUND: Inactivation of Shox2, a member of the short-stature homeobox gene family, leads to defective development of multiple organs and embryonic lethality as a result of cardiovascular defects, including bradycardia and severe hypoplastic sinoatrial node (SAN) and sinus valves, in mice. It has been demonstrated that Shox2 regulates a genetic network through the repression of Nkx2.5 to maintain the fate of the SAN cells. However, the functional mechanism of Shox2 protein as a transcriptional repressor on Nkx2.5 expression remains completely unknown. METHODS AND RESULTS: A specific interaction between the B56δ regulatory subunit of PP2A and Shox2a, the isoform that is expressed in the developing heart, was demonstrated by yeast 2-hybrid screen and coimmunoprecipitation. Western blotting and immunohistochemical assays further confirmed the presence of phosphorylated Shox2a (p-Shox2a) in cell culture as well as in the developing mouse and human SAN. Site-directed mutagenesis and in vitro kinase assays identified Ser92 and Ser110 as true phosphorylation sites and substrates of extracellular signal-regulated kinase 1 and 2. Despite that Shox2a and its phosphorylation mutants possessed similar transcriptional repressive activities in cell cultures when fused with Gal4 protein, the mutant forms exhibited a compromised repressive effect on the activity of the mouse Nkx2.5 promoter in cell cultures, indicating that phosphorylation is required for Shox2a to repress Nkx2.5 expression specifically. Transgenic expression of Shox2a, but not Shox2a-S92AS110A, mutant in the developing heart resulted in down-regulation of Nkx2.5 in wild-type mice and rescued the SAN defects in the Shox2 mutant background. Last, we demonstrated that elimination of both phosphorylation sites on Shox2a did not alter its nuclear location and dimerization, but depleted its capability to bind to the consensus sequences within the Nkx2.5 promoter region. CONCLUSIONS: Our studies reveal that phosphorylation is essential for Shox2a to repress Nkx2.5 expression during SAN development and differentiation.


Assuntos
Proteínas de Homeodomínio/fisiologia , Nó Sinoatrial/crescimento & desenvolvimento , Animais , Células Cultivadas , Ensaio de Desvio de Mobilidade Eletroforética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Frequência Cardíaca/fisiologia , Proteína Homeobox Nkx-2.5 , Humanos , Camundongos , Camundongos Transgênicos , Mutagênese Sítio-Dirigida/métodos , Fosforilação/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , Nó Sinoatrial/embriologia , Fatores de Transcrição/fisiologia , Técnicas do Sistema de Duplo-Híbrido
5.
Int Urol Nephrol ; 46(2): 341-7, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23982767

RESUMO

PURPOSE: To investigate the diagnostic performance of urinary brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) as potential biomarkers for overactive bladder (OAB). METHODS: Ninety women diagnosed with OAB and 45 normal controls without OAB were enrolled. Urine samples were collected from all subjects. Urinary BDNF and NGF levels were measured using enzyme-linked immunosorbent assays. Results normalized by urinary creatinine (Cr) levels were compared between OAB groups and controls. Symptom severity was assessed using overactive bladder symptom score. RESULTS: Urinary BDNF and NGF levels were elevated in OAB groups but not in controls. Mean (SD) baseline BDNF and NGF levels normalized by Cr levels were significantly higher in OAB subjects than in controls (20.609 ± 23.932 vs. 1.779 ± 0.729, p < 0.01) and (0.258 ± 0.264 vs. 0.081 ± 0.028, p < 0.01), respectively. Urinary BDNF/Cr levels were 80-fold higher than NGF/Cr levels in OAB subjects. Receiver operating characteristic curves for assessing urinary BDNF/Cr levels in OAB groups showed sensitivity and specificity of 93.33 and 88.89 %, respectively. Urinary BDNF levels were associated with OAB symptom severity. CONCLUSIONS: Urinary BDNF/Cr levels are elevated in women with OAB and are significantly associated with symptom severity. No elevation of BDNF is found in women without OAB. BDNF analysis has better sensitivity than NGF in detecting OAB in subjects without other lower urinary tract disorders. Results of the present study suggest a potential role for BDNF as an objective biomarker for OAB diagnosis.


Assuntos
Fator Neurotrófico Derivado do Encéfalo/urina , Fator de Crescimento Neural/urina , Bexiga Urinária Hiperativa/diagnóstico , Adulto , Biomarcadores/urina , Estudos de Casos e Controles , Creatinina/urina , Feminino , Humanos , Curva ROC , Índice de Gravidade de Doença
6.
Mol Biol Rep ; 39(3): 2781-7, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21681428

RESUMO

Chlorogenic acid (CGA) is one of the most abundant polyphenol compounds in human diet. It is also an active component in traditional Chinese medicines which are used to treat various diseases. In this study, fluorescence spectroscopy in combination with UV-Vis absorption spectroscopy was employed to investigate the specific binding of CGA to human serum albumin (HSA) under the physiological conditions. In the mechanism discussion, it was proved that the fluorescence quenching of HSA by CGA is a result of the formation of CGA-HSA complex. Binding parameters calculating from Stern-Volmer method and Scatchard method showed that CGA bind to HSA with the binding affinities of the order 10(4) l mol(-1). The thermodynamic parameters studies revealed that the binding was characterized by negative enthalpy and positive entropy changes and the electrostatic interactions play a major role for CGA-HSA association. Site marker competitive displacement experiments demonstrated that CGA specific bind to site I (subdomain IIA) of HSA. The binding distance r (3.10 nm) between donor (Trp-214) and acceptor (CGA) was obtained according to fluorescence resonance energy transfer. Furthermore, the effect of metal ions on CGA-HSA system was studied.


Assuntos
Ácido Clorogênico/metabolismo , Complexos Multiproteicos/metabolismo , Albumina Sérica/metabolismo , Transferência Ressonante de Energia de Fluorescência , Humanos , Ligação Proteica , Espectrometria de Fluorescência , Eletricidade Estática , Termodinâmica , Espectroscopia por Absorção de Raios X
7.
J Biol Chem ; 286(19): 17029-38, 2011 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-21454626

RESUMO

The homeodomain transcription factor Shox2 plays a crucial regulatory role in the development of sinoatrial node (SAN) by repressing the expression of Nkx2.5, as demonstrated by failed differentiation of SAN in Shox2 null mice. The SHOX (short stature homeobox) gene family consists of two closely related members, SHOX and SHOX2 in humans, but a SHOX ortholog does not exist in the mouse genome. These two genes exhibit overlapping and distinct expression patterns in many developing organs but whether they share functional redundancy is not known. In this study, we set to investigate possible functional redundancy between SHOX and SHOX2 in vitro and in vivo. We first showed that human SHOX and SHOX2 and mouse Shox2 possess similar transcriptional repressive activities in cell cultures, particularly the repressive effects on the Nkx2.5 promoter activity. We further created an SHOX/Shox2 knock-in mouse line (replacement of Shox2 with SHOX, referred as Shox2(KI/KI)). Mice carrying the hypomorphic Shox2(KI+Neo/KI+Neo) allele exhibit bradycardia and arrhythmia and die a few days after birth. However, mice carrying the Shox2(KI/KI) allele grow to adulthood. Physiological, histological, and molecular analyses demonstrate a fully developed SAN and normal pacemaking function in Shox2(KI/KI) mice. Our results demonstrate a functional redundancy between human SHOX and mouse Shox2 in the regulation of SAN formation and pacemaking function in addition to several other organs. The SHOX/Shox2 dose appears to be critical for normal pacemaking function.


Assuntos
Regulação da Expressão Gênica , Coração/fisiologia , Proteínas de Homeodomínio/metabolismo , Nó Sinoatrial/metabolismo , Alelos , Animais , Arritmias Cardíacas/metabolismo , Eletrocardiografia/métodos , Proteína Homeobox Nkx-2.5 , Proteínas de Homeodomínio/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Genéticos , Proteína de Homoeobox de Baixa Estatura , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
8.
Ai Zheng ; 25(6): 677-82, 2006 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-16764760

RESUMO

BACKGROUND & OBJECTIVE: The interaction between pro-apoptotic factors and anti-apoptotic factors is closely related to the genesis and development of tumors. Omi/HtrA2 is a novel gene involved in the regulation of apoptosis. PED/PEA-15 is a widely expressed anti-apoptotic protein. This study was to explore the effects of Omi/HtrA2 on PED/PEA-15 expression and apoptosis of prostate cancer cell line PC-3. METHODS: Omi/HtrA2 expression and specific siRNA vectors were constructed and transiently transfected into PC-3 cells. The effect of Omi/HtrA2 on PED/PEA-15 expression was assayed by Western blot, and its effect on apoptosis of PC-3 cells was analyzed by ELISA. Caspase-8 activity was assayed using Caspase-8 colorimetric assay kit. The effects of Omi/HtrA2-specific siRNA sequence on its transcription and translation were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. The sensitivity of PC-3 cells to cisplatin (DDP) after Omi/HtrA2 gene silencing was determined by flow cytometry. RESULTS: Enzyme digestion analysis and DNA sequencing confirmed Omi/HtrA2 expression, and specific siRNA vectors were successfully constructed. After transfection of Omi/HtrA2 expression vector, PED/PEA-15 expression was inhibited, Caspase-8 activity was promoted, and the apoptosis of PC-3 cells was enhanced. The sensitivity of PC-3 cells to DDP was suppressed after Omi/HtrA2 gene silencing. CONCLUSION: Omi/HtrA2 can promote the apoptosis of PC-3 cells through inhibiting PED/PEA-15 expression.


Assuntos
Apoptose , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Mitocondriais/genética , Fosfoproteínas/metabolismo , Neoplasias da Próstata/patologia , RNA Interferente Pequeno/genética , Serina Endopeptidases/genética , Antineoplásicos/farmacologia , Proteínas Reguladoras de Apoptose/metabolismo , Caspase 8/metabolismo , Linhagem Celular Tumoral , Cisplatino/farmacologia , Inativação Gênica , Vetores Genéticos , Serina Peptidase 2 de Requerimento de Alta Temperatura A , Humanos , Masculino , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/fisiologia , Plasmídeos , Neoplasias da Próstata/metabolismo , Serina Endopeptidases/metabolismo , Serina Endopeptidases/fisiologia , Transfecção
9.
Ai Zheng ; 23(1): 44-9, 2004 Jan.
Artigo em Chinês | MEDLINE | ID: mdl-14720373

RESUMO

BACKGROUND & OBJECTIVE: Helicobacter pylori (HP) is the important pathogen of chronic gastritis and peptic ulcer. HP infection is closely associated with extragastrointestinal disease of human and animals. This study was designed to investigate the effect of HP on hepatocarcinoma HepG2 cells. METHODS: HepG2 cells was cocultured with cagA (+) HP and cagA(-) HP at different concentrations. Then the cell morphology, DNA fragments electrophoresis, transmission electron microscopy, MTT method, and enzyme assays were used to observe the effect of HP on the morphology, growth rate, and enzyme change of HepG2 cells. RESULTS: (1) With the increasing concentrations of cagA (+) HP and prolongation of culture time, the morphology of HepG2 cells was changed from spindle shape to round; the ability of HepG2 cells adhering to wall were decreased; the ability of HepG2 cells in suspension were increased; and debris emitted around the cells; (2) With the increasing concentrations of cagA (+) HP, DNA ladder occurred; (3) With the increasing concentrations of cagA (+) HP and prolongation of culture time, the nuclei of HepG2 cells showed chromatin pyknosis, and clustered on the inner border of karyotheca, condensed cytoplasm with many vacuoles; (4) The MTT values of HepG2 cells were decreased with the increasing concentration of cagA(+) HP (P< 0.01); (5)The values of alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) in coculture supernatants of cagA (+) Hp and HepG2 were increased (P< 0.01); (6) No influence of cagA (-) Hp on HepG2 was found. CONCLUSIONS: cagA (+) Hp has in vitro cytotoxicity on HepG2 cells, and the cytotoxicity is positively related to the concentration and culture time of cagA(+) HP.


Assuntos
Carcinoma Hepatocelular/patologia , Helicobacter pylori/patogenicidade , Neoplasias Hepáticas/patologia , Alanina Transaminase/metabolismo , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Carcinoma Hepatocelular/ultraestrutura , DNA de Neoplasias/análise , Humanos , L-Lactato Desidrogenase/metabolismo , Neoplasias Hepáticas/ultraestrutura , Microscopia Eletrônica
10.
Eur J Hum Genet ; 11(12): 931-6, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-14508502

RESUMO

Maple syrup urine disease (MSUD) is an autosomal recessive inborn error disorder derived from the accumulation of the branched-chain amino acids (BCAAs) leucine, isoleucine and valine. Either the E1alpha, E1beta or DBT (E2) genes are responsible for this neurometabolic disease. Here, we report the identification and characterization of a novel E2 gene 4.7 kb deletion as a rare nonhomologous recombination of the long interspersed nuclear elements 1 (LINE-1) in intron 10 and the Alu in the 3' UTR of the E2 gene from three classic MSUD patients of the Austronesian aboriginal tribe Paiwan in Taiwan. The E2 gene 4.7 kb deletion accounted for five out of six alleles in the three unrelated Paiwanese MSUD patients, indicating a founder effect. Carrier-frequency study revealed one deleted heterozygote out of 101 normal Paiwanese. As the nine Taiwanese Austronesian aboriginal tribes share a common origin, this E2 4.7 kb deletion may be preserved in some of the other Austronesian aboriginal tribes of Taiwan. This is the first comprehensive genetics study of MSUD in the Austronesian tribal groups as well as in Taiwan.


Assuntos
Elementos Alu , Efeito Fundador , Triagem de Portadores Genéticos , Elementos Nucleotídeos Longos e Dispersos , Doença da Urina de Xarope de Bordo/genética , Grupo com Ancestrais Oceânicos , Sequência de Bases , DNA , Primers do DNA , Humanos , Dados de Sequência Molecular , Mutação , Deleção de Sequência , Homologia de Sequência do Ácido Nucleico , Taiwan
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